Quenching of G4-DNA intrinsic fluorescence by ligands.

G-quadruplex (G4) structures formed by the guanine-rich DNA regions exhibit several distinctive optical properties, including UV absorption and circular dichroism spectra. Some G4 DNA possess intrinsic UV fluorescence whose origin is not completely clear to date. In this work, we study the effect of TMPyP4 and Methylene Blue on the intrinsic fluorescence of the dimeric G4 DNA structure formed by two d(G3T)4 sequences. We demonstrate that binding of the ligands results in quenching of the intrinsic fluorescence, although the conformation of the G4 DNA and its dimeric structure remain preserved. The binding sites of the ligands were suggested by the photoinduced oxidation of guanines and analysis of binding isoterms. We discuss how DNA-ligand complexes can affect the intrinsic fluorescence of G4 DNA.

Taq-Polymerase Stop Assay to Determine Target Selectivity of G4 Ligands in Native Promoter Sequences of MYC, TERT, and KIT Oncogenes.

Computational and high-throughput experimental methods predict thousands of potential quadruplex sequences (PQSs) in the human genome. Often these PQSs contain more than four G-runs, which introduce additional uncertainty into the conformational polymorphism of the G4 DNA. G4-specific ligands, which are currently being actively developed as potential anticancer agents or tools for studying G4 structures in genomes, may preferentially bind to specific G4 structures over the others that can be potentially formed in the extended G-rich genomic region. We propose a simple technique that identifies the sequences that tend to form G4 in the presence of potassium ions or a specific ligand. Thermostable DNA Taq-polymerase stop assay can detect the preferential position of the G4 -ligand binging within a long PQS-rich genomic DNA fragment. This technique was tested for four G4 binders PDS, PhenDC3, Braco-19, and TMPyP4 at three promoter sequences of MYC, KIT, and TERT that contain several PQSs each. We demonstrate that the intensity of polymerase pausing reveals the preferential binding of a ligand to particular G4 structures within the promoter. However, the strength of the polymerase stop at a specific site does not always correlate with the ligand-induced thermodynamic stabilization of the corresponding G4 structure.

Differential Impact of Random GC Tetrad Binding and Chromatin Events on Transcriptional Inhibition by Olivomycin A.

Olivomycin A (OA), an antibiotic of the aureolic acid family, interferes with gene transcription upon forming complexes with GC-rich regions in the DNA minor groove. We demonstrate that the mechanism of transcriptional deregulation is not limited to OA interaction with GC-containing binding sites for transcription factors. Using electrophoretic mobility shift assays and DNAse I footprinting of cytomegalovirus (CMV) promoter fragments carrying OA-preferred GC tetrads (CMVwt), we showed OA binding specifically to GC islands. Replacement of G for A in these tetrads (CMVmut) abrogated OA binding. Furthermore, OA decreased RNA polymerase II (RNAPII) binding to the CMVwt promoter and inhibited the reporter gene expression. In line with the absence of OA binding sites in CMVmut DNA, the expression driven from this promoter was weakly sensitive to OA. In the endogenous genes OA decreased RNAPII on promoters and coding regions. In certain cases this phenomenon was concomitant with the increased histone 3 abundance. However, the sensitivity to OA did not correlate with GC patterns around transcription start sites, suggesting that certain GC stretches play unequal roles in OA-induced transcriptional perturbations. Thus, OA affects transcription via complex mechanisms in which GC tetranucleotide binding causes RNAPII/chromatin alterations differentially manifested in individual gene contexts.

Genome Assembly and Sex-Determining Region of Male and Female Populus × sibirica.

The genus Populus is presented by dioecious species, and it became a promising object to study the genetics of sex in plants. In this work, genomes of male and female Populus × sibirica individuals were sequenced for the first time. To achieve high-quality genome assemblies, we used Oxford Nanopore Technologies and Illumina platforms. A protocol for the isolation of long and pure DNA from young poplar leaves was developed, which enabled us to obtain 31 Gb (N50 = 21 kb) for the male poplar and 23 Gb (N50 = 24 kb) for the female one using the MinION sequencer. Genome assembly was performed with different tools, and Canu provided the most complete and accurate assemblies with a length of 818 Mb (N50 = 1.5 Mb) for the male poplar and 816 Mb (N50 = 0.5 Mb) for the female one. After polishing with Racon and Medaka (Nanopore reads) and then with POLCA (Illumina reads), assembly completeness was 98.45% (87.48% duplicated) for the male and 98.20% (76.77% duplicated) for the female according to BUSCO (benchmarking universal single-copy orthologs). A high proportion of duplicated BUSCO and the increased genome size (about 300 Mb above the expected) pointed at the separation of haplotypes in a large part of male and female genomes of P. × sibirica. Due to this, we were able to identify two haplotypes of the sex-determining region (SDR) in both assemblies; and one of these four SDR haplotypes, in the male genome, contained partial repeats of the ARR17 gene (Y haplotype), while the rest three did not (X haplotypes). The analysis of the male P. × sibirica SDR suggested that the Y haplotype originated from P. nigra, while the X haplotype is close to P. trichocarpa and P. balsamifera species. Moreover, we revealed a Populus-specific repeat that could be involved in translocation of the ARR17 gene or its part to the SDR of P. × sibirica and other Populus species. The obtained results expand our knowledge on SDR features in the genus Populus and poplar phylogeny.

Genome and Transcriptome Sequencing of Populus × sibirica Identified Sex-Associated Allele-Specific Expression of the CLC Gene.

Transcriptome sequencing of leaves, catkin axes, and flowers from male and female trees of Populus × sibirica and genome sequencing of the same plants were performed for the first time. The availability of both genome and transcriptome sequencing data enabled the identification of allele-specific expression. Such an analysis was performed for genes from the sex-determining region (SDR). P. × sibirica is an intersectional hybrid between species from sections Aigeiros (Populus nigra) and Tacamahaca (Populus laurifolia, Populus suaveolens, or Populus × moskoviensis); therefore, a significant number of heterozygous polymorphisms were identified in the SDR that allowed us to distinguish between alleles. In the SDR, both allelic variants of the TCP (T-complex protein 1 subunit gamma), CLC (Chloride channel protein CLC-c), and MET1 (DNA-methyltransferase 1) genes were expressed in females, while in males, two allelic variants were expressed for TCP and MET1 but only one allelic variant prevailed for CLC. Targeted sequencing of TCP, CLC, and MET1 regions on a representative set of trees confirmed the sex-associated allele-specific expression of the CLC gene in generative and vegetative tissues of P. × sibirica. Our study brings new knowledge on sex-associated differences in Populus species.

Recurrent Potential G-Quadruplex Sequences in Archaeal Genomes.

Evolutionary conservation or over-representation of the potential G-quadruplex sequences (PQS) in genomes are usually considered as a sign of the functional relevance of these sequences. However, uneven base distribution (GC-content) along the genome may along the genome may result in seeming abundance of PQSs over average in the genome. Apart from this, a number of other conserved functional signals that are encoded in the GC-rich genomic regions may inadvertently result in emergence of G-quadruplex compatible sequences. Here, we analyze the genomes of archaea focusing our search to repetitive PQS (rPQS) motifs within each organism. The probability of occurrence of several identical PQSs within a relatively short archaeal genome is low and, thus, the structure and genomic location of such rPQSs may become a direct indication of their functionality. We have found that the majority of the genomes of Methanomicrobiaceae family of archaea contained multiple copies of the interspersed highly similar PQSs. Short oligonucleotides corresponding to the rPQS formed the G-quadruplex (G4) structure in presence of potassium ions as demonstrated by circular dichroism (CD) and enzymatic probing. However, further analysis of the genomic context for the rPQS revealed a 10-12 nt cytosine-rich track adjacent to 3'-end of each rPQS. Synthetic DNA fragments that included the C-rich track tended to fold into alternative structures such as hairpin structure and antiparallel triplex that were in equilibrium with G4 structure depending on the presence of potassium ions in solution. Structural properties of the found repetitive sequences, their location in the genomes of archaea, and possible functions are discussed.

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug-DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA-DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

Tumor suppressor properties of the small C-terminal domain phosphatases in non-small cell lung cancer.

Non-Small Cell Lung Cancer (NSCLC) is responsible for the majority of deaths caused by cancer. Small C-terminal domain (CTD) phosphatases (SCP), CTDSP1, CTDSP2 and CTDSPL (CTDSPs) belong to SCP/CTDSP subfamily and are involved in many vital cellular processes and tumorigenesis. High similarity of their structures suggests similar functions. However their role in NSCLC remains insufficiently understood. For the first time we revealed the suppressor function of CTDSPs leading to a significant growth slowdown and senescence of A549 lung adenocarcinoma (ADC) cells in vitro. Their tumor-suppressive activity can be realized through increasing the proportion of the active form of Rb protein dephosphorylated at Ser807/811, Ser780, and Ser795 (P<0.05) thereby negatively regulating cancer cell proliferation. Moreover, we observed that a frequent (84%, 39/46) and highly concordant (Spearman's rank correlation coefficient (rs) = 0.53-0.62, P≤0.01) down-regulation of CTDSPs and RB1 is characteristic of primary NSCLC samples (n=46). A clear difference in their mRNA levels was found between lung ADCs with and without lymph node metastases, but not in squamous cell carcinomas (SCCs) (P≤0.05). Based on The Cancer Genome Atlas (TCGA) data and the results obtained using the CrossHub tool, we suggest that the well-known oncogenic cluster miR-96/182/183 could be a common expression regulator of CTDSPs. Indeed, according to our qPCR, the expression of CTDSPs negatively correlates with these miRs, but positively correlates with their intronic miR-26a/b. Our results reflect functional association of CTDSP1, CTDSP2, and CTDSPL, expand knowledge about their suppressor properties through Rb dephosphorylation and provide new insights into the regulation of NSCLC growth.

Pan-Cancer Analysis of TCGA Data Revealed Promising Reference Genes for qPCR Normalization.

Quantitative PCR (qPCR) remains the most widely used technique for gene expression evaluation. Obtaining reliable data using this method requires reference genes (RGs) with stable mRNA level under experimental conditions. This issue is especially crucial in cancer studies because each tumor has a unique molecular portrait. The Cancer Genome Atlas (TCGA) project provides RNA-Seq data for thousands of samples corresponding to dozens of cancers and presents the basis for assessment of the suitability of genes as reference ones for qPCR data normalization. Using TCGA RNA-Seq data and previously developed CrossHub tool, we evaluated mRNA level of 32 traditionally used RGs in 12 cancer types, including those of lung, breast, prostate, kidney, and colon. We developed an 11-component scoring system for the assessment of gene expression stability. Among the 32 genes, PUM1 was one of the most stably expressed in the majority of examined cancers, whereas GAPDH, which is widely used as a RG, showed significant mRNA level alterations in more than a half of cases. For each of 12 cancer types, we suggested a pair of genes that are the most suitable for use as reference ones. These genes are characterized by high expression stability and absence of correlation between their mRNA levels. Next, the scoring system was expanded with several features of a gene: mutation rate, number of transcript isoforms and pseudogenes, participation in cancer-related processes on the basis of Gene Ontology, and mentions in PubMed-indexed articles. All the genes covered by RNA-Seq data in TCGA were analyzed using the expanded scoring system that allowed us to reveal novel promising RGs for each examined cancer type and identify several "universal" pan-cancer RG candidates, including SF3A1, CIAO1, and SFRS4. The choice of RGs is the basis for precise gene expression evaluation by qPCR. Here, we suggested optimal pairs of traditionally used RGs for 12 cancer types and identified novel promising RGs that demonstrate high expression stability and other features of reliable and convenient RGs (high expression level, low mutation rate, non-involvement in cancer-related processes, single transcript isoform, and absence of pseudogenes).

Deep Sequencing Revealed a CpG Methylation Pattern Associated With ALDH1L1 Suppression in Breast Cancer.

Hypermethylation of promoter CpG islands is generally recognized epigenetic mechanism responsible for gene silencing in cancer. However, molecular details on how this epigenetic mark triggers the process of gene downregulation are still elusive. Here, we used deep bisulfite sequencing and qPCR analysis to investigate the pattern of CpG methylation of ALDH1L1 promoter region and its association with the gene expression level in 16 paired breast cancer (BC) samples of different clinical stages. Expression of ALDH1L1 gene was suppressed in all examined BC samples up to 200-fold, and average hypermethylation level of the promoter region correlated positively with ALDH1L1 downregulation. We determined the role of every individual CpG site within the ALDH1L1 promoter, including upstream untranscribed region, first untranslated exon, and the start of the first intron, in aberrant gene expression by correlation analysis. The search revealed CpG sites which methylation has the highest impact on intensity of gene transcription. The majority of such CpG sites are located in a compact region in the first intron of the ALDH1L1 gene. These results assist in unraveling of dynamic nature of CpG promoter hypermethylation as well as demonstrate the efficiency of deep bisulfite sequencing in search for novel epigenetic markers in cancer.

Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed 'short acid' (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models. We herein report that compound 3 is able to form stable complexes with DNA in the absence of Mg2+, in striking contrast to 1 whose binding to the DNA absolutely requires Mg2+. The mode of binding of 3 to DNA is similar in the presence or absence of Mg2+ as determined by circular dichroism. The affinity to DNA of 3 in Mg2+-free solution was similar to that of 1 or 3 in the presence of Mg2+ at low ionic strength. Non-electrostatic contributions to total free energy of binding of 1 and 3 to DNA were comparable for Mg2+-free complexes. Our data strongly suggest that electrostatic interaction of the positively charged 3 can compensate for the absence of divalent ions in complexes with DNA. This new property of the olivomycin A derivative expands the mechanistic knowledge of the modes of interaction with DNA of small molecular weight drug candidates.

Light-induced oxidation of the telomeric G4 DNA in complex with Zn(II) tetracarboxymethyl porphyrin.

Structure-specific ligands are convenient tools for the recognition, targeting or probing of non-canonical DNA structures. Porphyrin derivatives exhibit a preference for interaction with G-quadruplex (G4) structures over canonical duplex DNA and are able to cause photoinducible damage to nucleic acids. Here, we show that Zn(II) 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin ( ZNP1: ) interacts with different conformations of the telomeric sequence d(TAGGG(TTAGGG)3) at submicromolar concentrations without any detectible disturbance of the particular fold. Among different folds, potassium (3+1) hybrid G4-structure. reveal the highest affinity to ZNP1: The pattern of guanine oxidation is specific for each telomeric DNA conformation and may serve as an additional tool for probing the G4 topology. The potassium (3+1) and parallel G4 conformations are more susceptible to light-induced oxidation than the sodium G4 conformation or double helix of the telomeric DNA. The major products of the guanine modifications are spiroiminodihydantoin (Sp) and 8-oxoguanine (8-oxoG). ZNP1: -induced oxidation of guanines results in the structural rearrangement of parallel and (3+1) G4 conformations yielding an antiparallel-like G4 conformation. The mechanism of the observed light-induced conformational changes is discussed.

Identification of Novel Epigenetic Markers of Prostate Cancer by NotI-Microarray Analysis.

A significant need for reliable and accurate cancer diagnostics and prognosis compels the search for novel biomarkers that would be able to discriminate between indolent and aggressive tumors at the early stages of disease. The aim of this work was identification of potential diagnostic biomarkers for characterization of different types of prostate tumors. NotI-microarrays with 180 clones associated with chromosome 3 genes/loci were applied to determine genetic and epigenetic alterations in 33 prostate tumors. For 88 clones, aberrations were detected in more than 10% of tumors. The major types of alterations were DNA methylation and/or deletions. Frequent methylation of the discovered loci was confirmed by bisulfite sequencing on selective sampling of genes: FGF12, GATA2, and LMCD1. Three genes (BHLHE40, BCL6, and ITGA9) were tested for expression level alterations using qPCR, and downregulation associated with hypermethylation was shown in the majority of tumors. Based on these data, we proposed the set of potential biomarkers for detection of prostate cancer and discrimination between prostate tumors with different malignancy and aggressiveness: BHLHE40, FOXP1, LOC285205, ITGA9, CTDSPL, FGF12, LOC440944/SETD5, VHL, CLCN2, OSBPL10/ZNF860, LMCD1, FAM19A4, CAND2, MAP4, KY, and LRRC58. Moreover, we probabilistically estimated putative functional relations between the genes within each set using the network enrichment analysis.

2-aminopurine fluorescence: discrimination between specific and unspecific ligand binding to the kissing-loop dimer of the HIV-1 RNA.

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.

A protein component at the heart of an RNA machine: the importance of protein l27 for the function of the bacterial ribosome.

Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.